BIOCHEMICAL

Vol. 173, No. 3, 1990 December 31, 1990

AND BIOPHYSICAL

CLONING AND SEQUENCE ANALYSIS Yasuhiro Research

OF MINK GROWTH HORMONE cDNA

Hiroki Tatsumi,*Eiichi and Motoaki Umezu

Nakano

and Development Division, Kikkoman Corporation, 399 Noda, Noda-shi, Chiba 278, Japan Faculty of Agriculture, of Animal Reproduction, Tohoku University, Sendai 980, Japan

*Laboratory

Received

Harada,

RESEARCH COMMUNICATIONS Pages 1200-1204

November

13,

1990

A cDNA clone for mink growth hormone (GH) was isolated from a mink pituitary cDNA library, employing a part of rat growth hormone cDNA sequence as a probe. According to the nucleotide sequence, mature mink GH consists of 190 amino acids with a calculated molecular weight of 21,720. The amino acid sequence homology between the mature region of mink GH and those of pig GH, rat GH, bovine GH and human GH was 98.4%, 93.7%, 89.0% and 66.7%, respectively. "1990 Academic Press, Inc. SUMMARY:

Growth considered

hormone to

cently, goat

(3)

them

have

been

Mustela

vision,

GH (mGH) could growth

or

obtained

step

involved

GH genes

(Z),

lated

be

(GH) secreted

from

and

domestic

animals

(4)

of

mink

of

the

major

pituitary

quality.

mGH by means of MAmRIALS

coli

factor

In

nucleotide recombinant

Re-

(l),

pig

and

some of Mink,

(596).

for

However,

limited.

its

isolated,

is

growth.

as bovine

fur-producing

useful

is

pituitary

somatic

such

been

Escherichia

fur

anterior of

in

mGH cDNA and determined to produce

have

be a potential

improvement from

regulation

chicken

one

the

in

expressed is

from

animals.

Mink

stimulation

of

the this

amount study,

sequence,

of

GH

we isoas a first

DNA techniques.

AND METHODS

Bacterial strains and plasmids _E coli JM109 (ml,ii(b proAB),endAl,gyrA96,thi-l,hsdR17,relAl,supE44,[F'~36,proAB, was used as a host strain of recombinant plasmids. ~lacIqZAM151) Plasmid pUC118 and pUC119 were purchased from Takara Shuzo Japan) and used as cloning vectors. (Kyoto, 0006-291X/90 Copyright All rights

$1.50

0 1990 by Academic Press, of reproduction in any form

Inc. reserved.

1200

Vol. 173, No. 3, 1990

BIOCHEMICAL AND BIOPHYSICAL r~ESEARCH COMMUNICATIONS

Construction of eDNA library. Total RNA was isolated from fresh mink pituitaries obtained at Zao mink farm (Miyagi, Japan) with a hot phenol method described by Bancroft et al. (7), and double stranded eDNA was synthesized using a Pharmacia eDNA synthesis kit. Then, the eDNA was inserted into the SmaI site of pUCII9 (Takara Shuzo), followed by the transformation into E. coli JMI09, giving a eDNA library consisting of 1,500 independent clones. DNA sequencing Various subclones of mGH eDNA were constructed using the internal SmaI site and external restriction sites at the polylinker sequence of the vectors. The resultant plasmids were introduced into E. coli JMI01, respectively, to isolate single stranded DNA using bacteriophage MI3K07 (Takara Shuzo). DNA sequencing were performed by the dideoxy chain termination method described by Sanger et al (8). Both strands of the eDNA were completely determined by overlapping the SmaI site (Fig. i).

RESULTS

Cloning acid

of

a

mGH

sequences

sequence

mined.

of

of

hormone

eDNA mGH

mGH

(i0), Thus,

AND

clone. (9)

we

Analysis

revealed

completely

whose

DISCUSSION

that

synthesized

base

by

a

DNA

synthesizer.

probe,

we

performed

a

library

was

obtained,

nated eDNA

and

pMGHI00. cloned

On in

the

Using

colony

constructed

plasmid

restriction

regions

of

several

i0,ii),

we

considered

this

As

about GHs

that

this

bp

I

growth deter-

(TTCCCGGCCATG sequence

one this

for

bp

650

fragment

a

against

clone

(Fig.

that

as

positive

we

around

700

I

already

analysis,

Smal

t

been

acid

rat

experiment

in

were

0

of

result,

700

Pvull

I

a

enzyme

was

amino

that

DNA

amino

oligonucleotide

contained

mammalian

I

the

hybridization

above.

pMGHI00

had

of

region

partial

oligonucleotide

CCCTTGTCCAGCCTGTTTGCCAATGCCGTGCTC)

eDNA

with

sequence 45

the

N-terminal-15

coincided

nucleotide

of

was

found

i). bp

the strain desig-

that

I

coding

long

(1,2,3,

could

code

I

500bp

Fig. i. R e s t r i c t i o n map of the eDNA cloned in pMGHI00. The diagram shows the pre region (closed box), the mature region (open box) and the 3' n o n - c o d i n g region (dashed box). The sequencing strategy is indicated below the map. 1201

the

Since

Pvull

I

DNA

for

Vol. 173, No. 3, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the e n t i r e s t r u c t u r a l gene for mGH, and determined i t s n u c l e o t i d e

sequence.

Sequence a n a l y s i s of mGH cDNA.

Fig.

2 shows the n u c l e o t i d e

sequence of the cDNA and the amino acid sequence corresponding to the

l a r g e s t o p e n reading frame

t r u n c a t e d , while the 3'

(0RF). The 5'

end i s

end of the ORF i s

terminated by a TAG stop codon.

N - t e r m i n a l and i n t e r n a l amino a c i d sequences of mGH (9) found on the o p e n reading frame

(Fig.

2,

were

t h i c k and t h i n l i n e s ,

r e s p e c t i v e l y ) . These data suggest t h a t a cDNA for mature mGH has been c l o n e d , b u t

it

lacks N-terminal p o r t i o n for

the

signal

p e p t i d e . We are now p l a n n i n g to express t h i s cDNA fragment i n E. coil,

as

it

is

supposed to

c o n t a i n s u f f i c i e n t sequence

e x p r e s s i o n . From the n u c l e o t i d e sequence data,

it

for

the

i s deduced t h a t

mature mGH c o n s i s t s of 190 amino acids with a c a l c u l a t e d molecul a r weight of 21,720, which i s c o i n c i d e n t with t h a t determined by SDS-polyacrylamide

gel e l e c t r o p h o r e s l s (22,000).

e x t e n s i o n of 13 amino acids a t

The ORF has an

the N-terminal of mature r e g i o n ,

and other GHs from mammalian possess s i g n a l peptide c o n s i s t i n g of 26 amino a c i d s

( 1 , 2 , 1 0 , 1 1 ) . These f i n d i n g s i n d i c a t e t h a t

l e n g t h of t r u n c a t e d region i s around 13 amino a c i d s . The 3'

the non-

coding region i s 99 bp long and c o n t a i n s a consensus p o l y a d e n y l a t i o n s i g n a l , AATAAA, near the seems to be t r u n c a t e d .

3'

t e r m i n a l , but

a

poly(A)

tail

The experiment to i s o l a t e a f u l l l e n g t h

cDNA i s under going.

We c o m p a r e d with

those

Program (2), 93.7%,

of

amino

GHs f r o m

(Beckman),

rat

GH

89.0%,

(10), and

acid

sequence

various

and bovine 66.7%,

found

at

the

mammalians that

GH ( 1 ) , respectively 1202

mature using

homology and

human (Fig.

of GH 3).

region the

Micro

mGH w i t h (11)

are

of

mGH Genie

pig

GH

98.4%,

Vol. 173, No. 3, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CCCTGCTCAGCCTGCCCTGGCCTCAGGAGGTGGGCGCCTTCCCGGCCATGCCCTTGTCCA F P A M P L S ,, |, GCCTGTTTGCCAACGCCGTGCTCCGGGCCCAGCACCTGCACCAGCTGGCTGCCGACACCT S L F A N A V L R A Q H L H Q L A A D T

60

120

ACAAAGACTTTGAGCGGGCGTACATCCCCGAGGGCCAGAGGTACTCCATCCAGAACGCGC 1 8 0 Y K D F E R A Y I P E G Q R Y S I Q N A i

AGGCTGCCTTCTGCTTCTCGGAGACCATCCCGGCGCCCACCGGCAAGGACGAGGCCCAGC Q A A F C F S E T I P A P T G K D E A

Q

240

AGAGATCCGACATGGAGCTGCTCCGCTTCTCGCTGCTGCTCATCCAGTCGTGGCTGGGGC Q R S D M E L L R F S L L L I Q S W L

G

300

CCGTGCAGTTCCTCAGCAGGGTCTTCACCAACAGCCTGGTGTTCGGCACCTCGGACCGAG P V Q F L S R V F T N S L V F G T S D R

360

TCTACGAGAAGCTGAAGGACCTGGAGGAAGGCATCCAAGCGCTGATGCGGGAGCTGGAAG V Y E K L K D L E E G I Q A L M R E L E

420

ACGGCAGCCCCC GGGCCGGGCCGATCCTGAAGCAAACCTACGACAAGTTTGACACAAACC D G S P R A G P I L K Q T Y D K F D T N

480

TGCGCAGCGACGACGCGCTGCTCAAGAACTATGGGCTGCTCTC L R S D D A L L K N Y G L L

540

S

CTGCTTCAAGAAGGACC C F K K D

TGCACAAGGCCGAGACGTATCTGCGGGTCATGAAGTGTCGCCGCTTCGTGGAAAGCAGCT 6 0 0 L H K A E T Y L R V M K C R R F V E S S GTGCCTTCTAGTTGCCAGGCTTTCCTTTCACCCCGGCCCCACAGCCTCCCCTGACCCTGG C A F AAAGGGCCTCTCCCGGTGTCCACTGTGCTTTCCTAATAAAATGAAATTGG

660

710

Fig. 2. N u c l e o t i d e sequence of mGH. The nucleotide sequence is shown together with the predicted amino acid sequence of the largest ORF. The thick bar and the thin bars indicate the Nterminal and internal amino acid sequences of mGH, respectively, determined on Edman degradation.

m i n k GH p i g GH rat G H bovine GH h u m a n GH

48 -FPAMPLSSLFANAVLRAQHLHQLAADTYKDFERAYIPEGQRYSI-QNAQ E E A S G F E T T - TI R D M HR F QE E KE K FL P

m i n k GH p i ~ GH rat G H b o v i n e GH h u m a n GH

98 AAFCFSETIPAPTGKDEAQQRSDMELLRFSLLLIQSWLGPVQFLSRVFTN V E T I R V N K L I L TSL S T SNRE T K NL I E RS A

mink GH p i g GH rat GH bovine GH human GH

147 SLVFGTSD-RVYEKLKDLEEG I QALMRELEDGS PRAGPILKQTYDKFDTN Q M Q I Q A L T Q Y A SN DL T GR T QF S

m i n k GH p i g GH r a t GH b o v i n e GH h u m a n GH

190 LRSDDALLKNYGLLSCFKKDLHKAETYLRVMKCRRFVESSCAF M M SHN

Y

R R

T MD V

F

IVQ

A G A SG

G

Fig. 3. Comparison of amino acid sequences o f m i n k GH ( m G H ) , p i g GH, rat GH, bovine GH a n d human GH. The numbering is that of mature mGH. G a p s (-) were introduced to optimize the amino acid ali gnment. 1203

Vol. 173, No. 3, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES i) 2) 3)

4) 5) 6) 7) 8) 9) i0) ii)

Miller,W.L., Martial,J.A. and Baxter,J.D. (1980) J. Biol. Chem. 255, 7521-7524 Vize,P.D. and WelIs,J.R.E. (1987) Gene 55, 339-344 Yamano,Y., 0yabayashi,K., 0kuno,M., Yato,M., Kioka,N., Manabe,E., Hashi,H., Sakai,H., Komano,T., Utsumi, K. and Iritani,A. (1988) FEBS Lett. 228, 301-304 Lamb,I.C., Galehouse, D.M. and Foster,D.N. (1988) Nucleic Acids Res. 16, 9339 Seeburg,P.H., Sias,S., Adelman,J., De Boer,H.A., Hayflick,J., Jhurani,P., Goeddel,D.V. and Heyneker,H.L. DNA 2, 37-45 Vize,P.D. and WelIs,J.R.E. (1987) FEBS Lett. 213, 155-158 Bancroft,F.C., Wu,G.-J. and Zubay,G. (1973) Proc. Natl. Acad. Sci. USA 70, 3646-3649 Sanger,F., Nicklen,S. and Coulson,A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. Umezu,M. et al. in preparation Seeburg,P.H., Shine,J., Martial,J.A., Baxter J.D. and Goodman,H.M., (1977) Nature 270, 486-494 Martial,J.A., Hallewell,R.A., Baxter,J.D. and Goodman,H.M. (1979) Science 205, 602-606

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